Antiphospholipid antibodies: paradigm in transition.

OBJECTIVES: This is a critical review of anti-phospholipid antibodies (aPL). Most prior reviews focus on the aPL syndrome (APS), a thrombotic condition often marked by neurological disturbance. We bring to attention recent evidence that aPL may be equally relevant to non-thrombotic autoimmune conditions, notably, multiple sclerosis and ITP. ORGANIZATION: After a brief history, the recent proliferation of aPL target antigens is reviewed. The implication is that many more exist. Theories of aPL in thrombosis are then reviewed, concluding that all have merit but that aPL may have more diverse pathological consequences than now recognized. Next, conflicting results are explained by methodological differences. The lupus anticoagulant (LA) is then discussed. LA is the best predictor of thrombosis, but why this is true is not settled. Finally, aPL in non-thrombotic disorders is reviewed. CONCLUSION: The current paradigm of aPL holds that they are important in thrombosis, but they may have much wider clinical significance, possibly of special interest in neurology.

The effect of platelet activation on the hypercoagulability induced by murine monoclonal antiphospholipid antibodies.

BACKGROUND: To identify the mechanisms of the hypercoagulability associated with antiphospholipid antibodies, we investigated antibody-mediated platelet activation and interference of antibodies with phospholipid-dependent reactions. DESIGN AND METHODS: We used two murine monoclonal antibodies, one against beta(2)-glycoprotein I (7F6G), the other against prothrombin (28F4). Platelet activation was assessed by phospholipid-related platelet procoagulant activity. Endogenous thrombin potential without activated protein C (ETP(0)) and the activated protein C concentration that reduced the ETP(0) by 50% (IC(50)-APC) were determined by calibrated automated thrombography. RESULTS: Both monoclonal antibodies mimicked the effect of IgG in 11 out of a series of 40 patients with antiphospholipid antibodies in thrombography. In the presence of their target, 7F6G and 28F4 at 200 microg/mL exhibited comparatively low and high binding to platelets and elicited low and high levels of procoagulant phospholipids on platelet surface, respectively. In platelet-poor plasma, these antibodies induced a 1.6 and >12-fold increase in IC(50)-APC, respectively, thus providing evidence for a procoagulant effect independent of platelet activation. The 84% decrease in ETP(0) indicated that 28F4 also displayed an anticoagulant effect. In platelet-rich plasma, this anticoagulant effect was significantly less (23% decrease in ETP(0)), demonstrating that a high increase in procoagulant surfaces by platelet activation significantly antagonizes the anticoagulant effect of antiphospholipid antibodies. In both types of plasma, the inhibition of thrombin generation (reduced ETP(0)) was less than the inhibition of activated protein C activity (increased IC(50)-APC). CONCLUSIONS: Our findings show that platelet activation reinforces the hypercoagulability induced by competition between antiphospholipid antibodies/target complexes and pro- and anticoagulant complexes for phospholipid surfaces.

Laboratory evaluation of the antiphospholipid syndrome.

Antiphospholipid syndrome (APLS) is among the most common acquired blood protein defects that have been identified as leading to thrombosis. This article describes the laboratory diagnosis of APLS, including the detection of lupus anticoagulants, anticardiolipin antibodies, and subtypes of antiphospholipid antibodies.

Life-threatening bleeding in a patient with a lupus inhibitor and probable acquired factor VII deficiency.

We report the case of a 71-year-old man on warfarin for chronic atrial fibrillation presenting with a massive spontaneous soft tissue bleed. Despite reversing the effects of warfarin with large doses of intravenous vitamin K and fresh frozen plasma, bleeding continued, and his prothrombin time and activated partial thromboplastin time remained prolonged. The prothrombin time and activated partial thromboplastin time failed to correct with 50% normal plasma. Further investigations confirmed a lupus inhibitor with low levels of factors II, V, VII and XI. Factor II, V and XI levels normalized, however, when the patient's plasma was diluted 1:16 in buffer, suggesting the lupus inhibitor may have been interfering with these factor assays causing artefactual low results. Factor VII levels remained consistently low at all dilutions. The patient subsequently died following a massive left haemothorax despite surgical intervention and treatment with activated recombinant factor VII concentrate. We presumed the primary problem was bleeding from a local vascular lesion but the patient was never well enough to undergo confirmatory angiography. This case highlights the fact that patients with lupus inhibitors can develop severe haemorrhagic complications, and illustrates the complexities involved in both the investigation and treatment of abnormal bleeding in these patients.

Effect of anti-beta2glycoprotein I Lupus Anticoagulants on fibrin polymerization and fibrinolysis.

Anti-beta2-Glycoprotein I (beta2GPI) autoantibodies are the prominent laboratory feature of Hughes syndrome. By prolonging some coagulation tests in the presence of exogenous phospholipids (PL), they behave as classical Lupus Anticoagulants (LA). We investigated the effect of 3 affinity-purified anti-beta2GPI IgG preparations from patients with Hughes syndrome on fibrin polymerization and fibrinolysis of normal plasma, measured by comparing the optical densities of assay mixtures in the presence of the autoantibodies or normal IgG. The presence of anti-beta2GPI IgG in diluted Russell Viper Venom Time (dRVVT) assays, carried out using a PL dilution of 1:8 or 1:64, resulted in a delay in the onset of polymerization by 30-40 and 60-70s, respectively. Fibrin polymerization was complete after 250s for both anti-beta2GPI IgG and normal IgG. The inhibitory effect of the anti-beta2GPI antibodies was not observed in the presence of excess PL, as expected for LA. Anti-beta2GPI IgG increased the plateau level of polymerization when dRVVT was performed in the presence of 1.5 nM recombinant tissue plasminogen activator, but did not impair the fibrinolytic process, which was almost complete after 250 min. The autoantibodies did not delay the onset of fibrin polymerization in tests carried out using recombinant tissue factor. On the contrary, the autoantibodies enhanced polymerization in prothrombin time assays, and accelerated it in tissue thromboplastin inhibition tests, with no effect on fibrinolysis. These data provide evidence that anti-beta2GPI LA may act as either anticoagulants or procoagulants in different in vitro coagulation tests.

The effect of lupus anticoagulant in the second-generation assay for activated protein C resistance.

The activated protein C resistance (APCR) assay is the test of choice to screen for factor V Leiden. We evaluated the effect of lupus anticoagulant on the baseline clotting time of the second-generation APCR assay with plasma samples from 54 patients to determine whether a falsely low APCR ratio could be predicted. We also assessed whether a modification of the assay could make it more reliable in the presence of strong lupus anticoagulants. Of 54 plasma samples, 5 yielded a false-positive APCR ratio, and all 5 had a prolonged baseline clotting time. Further dilution (1:40) of the plasma samples in factor V-deficient plasma led to correction of the APCR ratio and did not affect the sensitivity of the test for factor V Leiden. Our data support that the baseline clotting time is a good predictor of a false-positive APCR test result and should be checked before calculating the ratio. The modified APCR assay reliably identified the false-positive ratios and could be used to screen for factor V Leiden in samples with strong lupus anticoagulant.

Improved characteristics of aPC-resistance assay: Coatest aPC resistance by predilution of samples with factor V deficient plasma.

Screening for a resistance against activated protein C (aPCR), which is in most cases caused by FV:Q506 mutation, is performed by functional tests measuring the effect of aPC on activated partial thromboplastin time (aPTT). Because of an insufficient discrimination between FV:Q506 mutation negative and positive individuals with the first generation of the functional test Coatest aPC Resistance (Chromogenix AB, Mölndal, Sweden), the definition of an arbitrary cut-off level was only possible using the results of DNA analysis. The use of an arbitrary cut-off level still resulted in unsatisfactory low sensitivity and specificity for the functional test. Thus, time- and cost-consuming DNA analyses had to be performed frequently to establish the diagnosis. The objective of this study was to evaluate an improved version of this assay that uses predilution of samples with factor V deficient plasma containing a heparin neutralizer. Using the data from 32 FV:Q506 mutation positive and 55 mutation negative individuals, the authors calculated a cut-off value resulting in an enhanced sensitivity (0.91 versus 1.0) and specificity (0.77 versus 1.0) compared to the old one. Imprecision was lowered from 5.36% (first generation) to 2.43%, in particular in samples with longer clotting times. In patients with prolonged aPTT, either caused by therapy with oral anticoagulants or heparin, correct results were obtained with the second generation assay, in contrast to the first generation assay. With this second generation assay the number of DNA analyses can be substantially reduced.

The impact of oral anticoagulant therapy, factor VIII level and quality of factor V-deficient plasma on three commercial methods for activated protein C resistance.

Several methods are now available for the laboratory assessment of activated protein C resistance (APCR). In this study, we evaluated two activated partial thromboplastin time-based assays [Coatest activated protein C (APC) and Diagen protein C activator (PCA)], with and without predilution of test plasma in factor V-deficient plasma (FVdp) and an amidolytic assay (Immuno Ltd, Vienna, Austria). Testing plasmas from normal volunteers who had received 1-deamino-8-D-arginine vasopressin (DDAVP) also assessed the effect of elevated factor VIII on APCR. In the unmodified clotting tests, the Coatest kit gave overlapping results for normal and heterozygous FV:Q506 samples; some FV:Q506 samples on oral anticoagulant therapy (OAT) were misclassified as normal, and some normal samples with high factor VIII levels would be classified as APC resistant. The unmodified Diagen kit correctly classified these three types of sample, but had the disadvantage that prolonged PCA clotting times gave serious problems with instrument end-point detection. Both kits modified by diluting the samples in FVdp correctly classified all the samples, as well as samples from patients with lupus anticoagulant (LA) and patients receiving heparin. The Immunochrom kit correctly classified the normal and FV:Q506 samples, but would have misclassified most normal persons on OAT as well as some patients with LA or receiving heparin therapy as APC resistant.

The role of beta 2-glycoprotein I-dependent lupus anticoagulants in the pathogenesis of the antiphospholipid syndrome.

The antiphospholipid syndrome (APS) is defined as the association of antiphospholipid antibodies (aPL) with arterial or venous thrombosis, recurrent fetal loss, thrombocytopenia or neurologic disorders. Some aPL can be detected via phospholipid dependent coagulation assays where they present as an aspecific coagulation inhibitor termed the lupus anticoagulant (LA). Other antibodies can be measured via immunological assays mostly via their capability to bind to immobilised cardiolipin and are therefore called anticardiolipin antibodies (aCL). Affinity purification of aCL led to the discovery that, in contrast to what the term antiphospholipid antibody could suggest, these autoimmune antibodies do not bind to negatively charged phospholipids per se but to beta-2-glycoprotein I (beta 2GPI), a phospholipid-binding protein eventually bound to phospholipid surfaces. LAs have been found to be directed towards either prothrombin or beta 2GPI bound to anionic phospholipids. Whereas clinical and animal experimental data clearly suggest a role for beta 2GPI-dependent aPL in the development of the APS, the pathogenic mechanism is not known. Interferences with several phospholipid dependent anticoagulant pathways have been proposed but none of these has received general acceptance. Based on clinical and experimental similarities with heparin-induced thrombocytopenia, another syndrome of antibody mediated thrombosis, we proposed a model of prothrombotic cellular activation. This model, although supported by a number of experimental observations, does not provide a direct explanation for the recent observation that LA are more strongly associated with thrombosis than aCL. In order to study this, we raised murine monoclonal antibodies (moab) against human beta 2GPI. These antibodies, of which some had LA activities and others not, enabled us to study the interaction between beta 2GPI, antibody and phospholipids. In contrast to what was generally accepted, beta 2GPI appeared to have only low affinity for coagulation promoting phospholipids. In the presence of LA positive anti-beta 2GPI moabs, the affinity of beta 2GPI for phospholipids increased significantly. This appeared to be dependent on the formation of bivalent beta 2GPI-antibody complexes on the phospholipid surface. It is conceivable that such bivalent complexes also remain tightly attached to membranes of activated cells enabling further thrombosis promoting activation via Fc receptor interaction or the complement system, a hypothesis that is currently being investigated. Further studies also showed that our LA positive anti-beta 2GPI moabs have a potential for the production of LA control specimens, that could be made available to routine hemostasis laboratories to assess intra-laboratory precision of LA testing, to manufacturers to produce highly sensitive assay systems and to control batch-to-batch variability of their reagents and to organizations involved in external quality assessment. In conclusion this work has enabled us to understand the molecular mechanism by which certain autoimmune antibodies found in patients with APS prolong coagulation assays in vitro. The antibodies generated are an important tool to improve the laboratory diagnosis of the lupus anticoagulant and may help us clarify the pathogenic role of autoimmune anti-beta 2GPI antibodies.

Transient lupus anticoagulant: an unusual cause of bruising in children.

A child presented with excessive bruising and prolonged activated partial thromboplastin time. Mixing studies in plasma were positive for phospholipid dependence of the anticoagulant, confirming a diagnosis of lupus anticoagulant. Factor II level was reduced. Laboratory findings normalised after three months, with spontaneous resolution of bruising. This case demonstrates a transient antiphospholipid antibody syndrome as a rare presentation of bleeding diathesis in a previously healthy child, and should be considered in children with new onset bruising and prolonged activated partial thromboplastin time.

Falsely elevated INRs in warfarin-treated patients with the lupus anticoagulant.

The Lupus Anticoagulant (L.A.) is an antibody that prolongs the clotting time of in-vitro laboratory tests by binding phospholipid in the test system. Patients with the L.A. are at increased risk for development of venous and arterial thrombosis but not hemorrhage. Therefore, many patients with the L.A. are being treated with warfarin sodium to prevent reoccurrence of thrombosis. This oral anticoagulant therapy is traditionally regulated by periodic determination of the Prothrombin Time (PT). This test is usually unaffected by the L.A. However, we have recently identified a small series of patients with the L.A. in whom the PT is affected by the L.A. This interference is manifest as an artifactually increased International Normalized Ratio (INR). These patients were identified by failure to achieve significant correction of the PT with addition of an equal volume of normal plasma to the patient plasma and a Factor X level discordant with the PT INR Interference in determination of the PT by the L.A. was found to occur in 6.5% of patients identified with the L.A. by our laboratory. It is suggested that patients with this complication of anticoagulant therapy be monitored by measurement of Factor X levels rather than the PT INR. Failure to recognize this complication may result in inadequate anticoagulation and recurrent thrombosis.

[Constitutional thrombophilias: indications of the biological profile and therapeutic consequences]. / Thrombophilies constitutionnelles: indications du bilan biologique et conséquences thérapeutiques.

In laboratory screening in patients with clinical thrombophilia (early thromboembolism episode < 50 years, spontaneous thrombosis, recurrent thrombosis, unusual site of thrombosis, thrombotic family history or coumarin-induced skin necrosis complication), an isolated or combined inherited thrombophilia can be observed: antithrombin (0.5 to 4.9 per cent), protein C (1.4 to 8.6 per cent) and protein S (1.4 to 7.5 per cent) deficiencies or factor V Leiden (20 to 30 per cent). Special attention is mandatory in prescribing biological exploration because of the many physiological or pharmacological interferences which can modify the results. Identification of a genetic defect may induce specific management and individuals should receive counselling regarding the implications of this diagnosis. Further prospective studies should help to determine the thrombotic risk in symptomatic and non-symptomatic patients with inherited thrombophilia and the risk/benefit ratio of laboratory screening for hereditary thrombophilia and therapeutic intervention.

[Association between antiphospholipid antibodies and arterial or venous thrombosis/thrombocytopenia].

The relationship between thrombotic or thrombocytopenic complications and the existence of anticardiolipin antibodies (aCL) and/or lupus anticoagulant (LA) was studied in 146 patients with systemic lupus erythematosus (SLE). The prevalence of arterial thrombosis was obviously higher in patients who had both aCL and LA than in patients with either aCL or LA alone or in those with neither. Since a substantial fraction of the former group of patients with arterial thrombosis also had thrombocytopenia, there is a possibility that aCL and LA might enhance platelet activation and aggregation. To test this hypothesis, we studied the in vitro effects of aCL and LA on the enhancement of platelet activation by flow cytometric analysis using anti-CD62P and anti-CD41 monoclonal antibodies directed against platelet activation-dependent granule-external membrane (PADGEM) protein and platelet glycoprotein IIb (GPIIb). The IgG fraction purified from aCL+.LA+ plasma apparently enhanced platelet activation induced by adenosine diphosphate (ADP) at a low concentration, but IgG fractions from aCL+.LA- or aCL-.LA+ plasma did not cause enhancement of platelet activation. These results suggest that aCL and LA may cooperate to promote platelet activation, and may be involved, at least partially, in the pathogenesis of arterial thrombosis in patients with SLE.

[The effects of lupus anticoagulant on pulmonary thromboembolism].

OBJECTIVE: To evaluate the effects of lupus anticoagulant (LA) on pulmonary thromboembolism (PTE). METHODS: Thirty-eight patients with PTE (17 massive and 21 submassive) and 30 healthy adults were studied. Russell's viper venom time (RVVT) was used to examine the ratio of LA (LAR), and a colorimetric method was used to detect the activity of plasma protein C (PC:A) and radioimmunoassay (RIA) was employed to measure the level of plasma thromboxane B(2) (TXB(2)) and 6-keto-prostaglandin F1alpha (6-keto-PGF1alpha). RESULTS: Compared with the normal group, LAR, TXB(2) and TXB(2)/6-keto-PGF1alpha showed significant increase in the massive PTE and the submassive PTE groups (P < 0.01), and the levels were higher in the massive group than in the submassive group (P < 0.01). Both groups showed significant decrease in PC:A and 6-keto-PGF1alpha compared with the normal group (P < 0.01). CONCLUSIONS: LA can increase TXB(2)/6-keto-PGF1alpha and decrease PC:A in patients with PTE. It is suggested that there may be an association between the increase of LAR and the presence of PTE.